Optimisation of a coupled enzymatic assay of glutathione peroxidase activity in bovine milk and whey

Glutathione peroxidase (GHSPx) may have important functions in mirk but studies of its activity during dairy processing are hampered by the lack of suitable assays. Thus, a coupled enzymatic assay of GHSPx activity was developed for bovine milk and whey. The reaction mixture contained reduced glutathione, tert-butylhydroperoxide, glutathione reductase and NADPH in phosphate buffer. The effect of reaction conditions on enzymatic and non-enzymatic NADPH consumption was studied, and blanks without added GHSPx sample or complete incubations containing 4 mmol L-1 mercaptosuccinate as an inhibitor of GHSPx were used. With increasing pH and glutathione concentration the reaction rates increased both in the complete incubation and the blank but optimal GHSPx activity was attained at a glutathione concentration of 0.63 mmol L-1. A pH of 7.6 was chosen to attain near-optimal activity without reaching high blanks. Application of the new procedure showed that bulk whey samples had rather constant GHSPx activity with an average (SD) of 36.1 (2.9) U mL(-1), but whey samples prepared from individual cows showed a two-fold Variation (range 24.5-50.6 U mL(-1)). The procedure described was more reproducible and precise than previous methods and will be employed in further studies of dairy products. (C) 2000 Elsevier Science Ltd. All rights reserved.